This proposal tests the hypothesis that herpes simplex virus type I (HSV-1) codes for and expresses specific gene(s) responsible for killing cells. These sequences will be identified by testing their ability to reduce the frequency of DNA-mediated gene transformation of Ltk- neomycin-sensitive cells to a thymidine kinase positive or neomycin resistant phenotype. The assumption is that cells will take up anti-transformation (A.T.) or "cell killing" sequences with the selectable marker by co-transformation and will die. Preliminary evidence suggests that co-transformation of Bgl 2-, or PVU-2-or Bam Hl- digested HSV-1 DNA with the purified HSV tk gene does not reduce the frequency of DNA mediated gene transfer of Ltk cells. Full length, EcoR-1, Xba 1- or Hind III-digested DNA does. Therefore, it appears that EcoR-1, Xba 1, and Hind III may not cleave within "cell killing" genes and isolated restriction fragments can then be tested for "anti-transformation" activity. Further studies to confirm the cytotoxicity of "anti-transformation" sequences are planned. Ltk-cells microinjected with individual or combinations of cloned restriction fragments will be tested for viability. In addition, mutagenized sequences will be "marker-transferred" to wild type HSV-1 and recombinants will be isolated and tested for altered cell killing ability. The gene products encoded by the anti-transformation sequences will be determined by positive mRNA selection and in vitro protein synthesis. Attempts to isolate transformants which express A.T. sequences will be made. Such transformants may be useful in establishing the functional relevancy of the A.T. region by examining the cells behavior following virus infection. Development of this transformation inhibition assay should allow us to propose specific viral sequences and functions as candidate cell killing elements. This will have important clinical as well as basic science interest. Finally, pilot studies suggest a particular EcoR-1 fragment does inhibit transformation.